The overall goal of this project is to characterize a novel cell death pathway, termed "aponecrosis", and determine its role in neurodegeneration. Extensive evidence was generated in recent years suggesting that two "classic" cell death pathways, apoptosis and necrosis, do not explain the variety of physiological and pathological cell death mechanisms. Existence of the third pathway, termed "aponecrosis", is proposed. This pathway is activated in cells that are induced to undergo apoptosis, yet prevented from its completion. It is proposed to represent a novel safety mechanism aimed at elimination of damaged and potentially dangerous cells. Aponecrosis shares programmed cell death nature with apoptosis and execution subroutines and phenotypic features with necrosis. Using high throughput screening of small molecule library several chemical inhibitors of aponecrosis were isolated. These compounds were found to selectively inhibit aponecrosis, but not apoptosis, underscoring distinct mechanism of the aponecrotic cell death process. Using selected inhibitors aponecrosis was shown to represent a major cell death process in a variety of caspase-independent cell death paradigms in vitro. Aponecrosis was also implicated in amyloid-beta toxicity in PC12 cells and ischemic brain damage in vivo. Analysis of the effects of selected inhibitors in various systems suggests that two of them target cell type specific signaling pathways, whereas another compound blocks uniform downstream execution step, providing the opportunity to characterize various steps in aponecrosis with the help of individual inhibitors. Two aponecrotic regulators (p38 kinase and cathepsin B) were discovered. Specific aims of this study are as follows: 1 ) To evaluate the role of aponecrosis in neuronal cell death in vitro and in vivo; 2) To characterize molecular events involved in aponecrosis; 3) To identify novel genes involved in aponecrosis using chemical, cell and molecular biology approaches. The research will be conducted in the Department of Cell Biology at Harvard Medical School under the joint guidance of Dr. Junying Yuan and Dr. Timothy Mitchison. Dr. Junying Yuan is a recognized leader in the field of neuronal cell death. Dr. Timothy Mitchison is an outstanding cell biologist, who is also one of the pioneers of chemical biology approach. Dr. Yuan will provide resources and mentorship for the first two and partially for the third aim. Dr. Timothy Mitchison will provide training and supervision as well as resources of Harvard Institute of Chemistry and Cell Biology for the chemical biology part of the project. Department of Cell Biology is fully committed to the applicant's development into a fully independent researcher in the field of aging-related neurodegeneration. Dr. Alexei Degterev is aspiring to become a fully independent research scientist in the field of aging-associated neuronal cell death and would use K01 training period to develop and enhance the skills needed to perform independent research in this area and develop research project that will from the basis for his future independent research.